Ezview red anti ha affinity matrix

Cells were grown on YPD to an OD₆₀₀ of 1.5–2.5 and switched to methanol media. For all the co-IPs, ∼350 OD₆₀₀ unit cells were used. Cells were resuspended in 2 ml IP-lysis buffer (20 mM Hepes-KOH, pH 7.4, 0.15 M NaCl, 1% CHAPS, 5 mM NaF, 50 µg/ml leupeptin, 50 µg/ml aprotinin, 1 mM PMSF, and yeast PIC with 20 mM EDTA, pH 8.0). Cells were lysed by vortexing with acid-washed glass beads. Lysate was then solubilized for an hour at 4°C with rotation. The lysate was centrifuged at 20,000 g for 20 min. For Pex19-FLAG co-IP (Fig. 6), cells were lysed in IP-lysis buffer without CHAPS, and the lysate was spun at 20,000 g. The supernatant was removed, and the pellet of the 20,000 g spin (20P) was solubilized with IP-lysis buffer with 1% CHAPS. The lysate was incubated with specified antibody affinity matrix (EZview Red Anti-HA Affinity matrix [E6779; Sigma-Aldrich] for IP of Pex17-3HA and Pex2-3HA; EZview Red Anti-FLAG M2 Affinity matrix [F2426; Sigma-Aldrich] for Pex19-FLAG). Lysates were incubated for 3 h at 4°C. The beads were then washed 5× with IP lysis buffer, and proteins were eluted with the addition of 150 µl of 1× nonreducing sample buffer and heating at 65°C. The eluate was analyzed by SDS-PAGE followed by Western blotting with specified antibodies.